**1. Introduction**

Olive-mill wastewaters (OMWs), the major e ffluent deriving from the olive oil production process, are considered as one of the most challenging agro-industrial wastes to treat, since they are produced in very high quantities. Moreover, their strong odor and dark color and their relatively high organic load have a direct negative impact on the environment if they are released untreated. This important residue of the olive oil industry is one of the most di fficult to treat wastes because of its high content in phenolic compounds [1,2]. The increased concentration of OMWs (without prior treatment) in organic matter and phenolic components results in the reduction of the available concentration of oxygen to the organisms. Lack of oxygen to the organisms upsets the balance of ecosystems and the soil porosity, resulting in contaminated aquifers and polluted environments [1,3,4]. Consequent problems are the production of odors, the excessive growth of algae and bacteria, and the appearance of the eutrophication phenomena in aquatic environments in which OMWs are released untreated. The organic matter in OMW consists of sugars, phenols, tannins, polyphenols, aromatic molecules, ash, and, in some cases, lipids and nitrogen content [2]. The phenolic compounds are mainly considered responsible for the toxicity of the OMWs constituting, in several cases, the limiting step of their large-scale managemen<sup>t</sup> [5–8]. It should be noted that, in several cases (i.e. in the case where traditional press extraction process is applied in order to liberate olive oil), OMWs containing very high concentrations of sugars (mostly glucose, in concentrations ≥65 g/<sup>L</sup> [2]) are simultaneously liberated. However, a new trend that has appeared in relation to the valorization of these wastewaters refers to their simultaneous utilization as a substrate and water treatment in various fermentation processes. Value-added metabolites could be produced during these fermentation processes with simultaneous partial detoxification (i.e. decolorization and phenol removal) of the residue [2–4,9–11].

The continuous development of biological liquid fuel industry (principally that of biodiesel and bioethanol production) makes mandatory the process and exploitation of the increased production of its main by-product and principal liquid residue, which is the concentrated glycerol-containing water called crude glycerol (crude glycerin) [12]. Specifically concerning biodiesel production facilities, the synthesis of 10.0 kg of biodiesel deriving from trans-esterification of various oils, generates *c.* 1.0 kg of glycerol (purity ≈90.0% *w*/*w*) [13,14]. Global biodiesel production has been growing in recent years. In 2016 more than 30.8 × 10<sup>6</sup> t. were produced (7.5% more than in 2015), and it is estimated that more than 3.0 × 10<sup>6</sup> t. of glycerol as an important industrial byproduct were produced only from biodiesel manufacture [15]. Biodiesel-derived glycerol is a grea<sup>t</sup> substrate for microbial growth due to its high concentration in carbon and inorganic constituents. Many studies have reported the ability of eukaryotic microorganisms to convert glycerol into a plethora of value-added compounds, such us microbial lipids (single cell oils; SCOs) [16–25], citric acid [11,26–32] polyols [20,33–35], enzymes, and microbial mass [13,14,20,28,29,32,34,36–44]. Biodiesel-derived glycerol as a carbon source employed in the Industrial Microbiology has many advantages over other conventional substrates frequently used as microbial substrates (i.e. commercial sugars). One such advantage is the significantly low (or even negative) acquisition cost that this renewable material presents. In spite of the fact that the industrial production of biodiesel is currently being accompanied by the side production of relatively highly purified industrial glycerol feedstock (e.g. purity of ≈85–90% *<sup>w</sup>*/*<sup>w</sup>*, sometimes >90% *w*/*w*), which can be used directly as the starting material for the chemical synthesis of epichlorohydrin, or it can be used in various pharmaceutical applications after further purification processes. The acquisition cost of this relatively highly purified feedstock (i.e. purity ≈85–90% *w*/*w*) is significantly low, and at the beginning of 2016, in the Asiatic markets, it was at *c*. 0.16 US \$/kg, with a systematic decreasing trend. Moreover, in several cases, a large fraction of low-quality glycerol deriving from biodiesel production process, the so-called "pitch" glycerol, is a typical waste-stream of the process amenable only for incineration, adding to climate relevant emissions (CO2 as well as N2O) and to the cost of the whole biodiesel production chain. Moreover, utilization of glycerol as microbial substrate is not competitive with substrates that can be used as edible products. It also has inorganic components such as potassium, calcium, sulfur and magnesium that can work favorably for microbial growth [45].

*Y. lipolytica* yeasts are ideal candidates for the remediation and valorization of OMWs as well as of other recalcitrant wastes and residues due to their ability to vigorously grow on a variety of substrates and to tolerate usually hostile growth media [46]. Moreover, *Y. lipolytica* yeasts have been reported to be capable of producing SCO having equivalent synthesis and value to that of cocoa-butter [47–50] and a plethora of other value-added compounds during growth on several types of agro-industrial co-products and residues of either fatty or hydrophilic chemical structures [4,36,44,51,52]. The present study investigated the ability of *Y. lipolytica* strain ACA-YC 5031, a strain that has been reported capable of producing SCO and citric acid during growth on glucose-based media under nitrogen-limited conditions [53], to grow and produce secondary metabolites on mixed nitrogen-limited media consisting of OMWs and biodiesel-derived glycerol. In fact, OMWs were used in order to partially replace tap water from the fermentations performed, whereas the e ffect of the addition of di fferent sodium chloride (NaCl) concentrations upon the physiological behavior of the strain on osmotic stress over the production of secondary metabolites was assessed. The number of investigations dealing with the cultivation of microorganisms on media that are composed of mixtures of OMWs with other low-cost substrates (i.e. commercial-type glucose, low-cost oils, etc.), in the approach in which OMWs are considered as simultaneous substrate and process water, are relatively restricted in the literature [3,8–11]. Moreover, studies in which blends of glycerol and OMWs as substrates were used are indeed very scarce [4]. On the other hand, the simultaneous e ffect of the addition of OMWs and NaCl upon the fermentation of glycerol by *Y. lipolytica* strain, to the best of our knowledge, is studied for the first time in the literature. Therefore, the potential of a new natural *Y. lipolytica* strain (ACA-YC 5031) to reduce the color and the phenol content and simultaneously to convert the industrial wastes (OMWs blended with glycerol and NaCl) into value-added compounds, useful in Industrial and Food Biotechnology, was investigated, and technological approaches of the bioprocess were critically addressed and discussed.
